1. Macrophages play an active role in orthodontics.
To determine the changes in the macrophages during orthodontics, a set of publicly available ScRNAseq data was analyzed. Sequencing data are derived from the alveolar bone of normal or orthodontically-treated mice. Data were subjected to basic bioinformatics analysis followed by quality control and annotated into 11 clusters based on the MouseRNAseqData dataset (Fig. 1A). The orthodontic group consisted of 2056 cells, and the control group consisted of 2845 cells. Next, we focused on the differences in the macrophages between two groups (Fig. 1B); 837 macrophages and 1352 macrophages were observed in the orthodontic and control groups, respectively. Dimensionality reduction was performed on macrophage subsets and differentially expressed genes (DEGs) were identified using the FindAllMarkers() function (Fig. 1C). Heatmaps and violin plots revealed the DEGs in two groups (Fig. 1C, D). The macrophages in the orthodontic group highly expressed genes that regulate bone metabolism, immune regulation, and angiogenesis, indicating that they could be involved in regulating the microenvironment during OTM and improving bone remodeling. Subsequently, Gene Ontology(GO) enrichment was performed based on the DEGs (Fig. 1E, F). The macrophages in the orthodontic group demonstrated higher levels of bone remodeling, immune response, and other functions. In addition, unsupervised single-cell trajectories were constructed using Monocole, and significant differences in macrophage gene expression levels between orthodontic and control groups were observed (Fig. 1G, H, S1). These findings suggested that macrophages might improve the efficiency of OTM by regulating the microenvironment.
2. The inflammatory environment affects the efficiency of OTM.
A rat OTM model was established by applying a mechanical device in the rat's mouth for 10 days (Fig. 2A). The rats were randomly divided into four groups as follows: control group (no treatment); orthodontic group (O group, wherein the maxillary first molars were orthodontically moved for ten days); orthodontic + periodontitis group (OP group, the model was induced by tying a wire on the maxillary first molar and applying a tensile force to the maxillary first molar for 10 days); and orthodontic after periodontitis treatment group (OPL group, in which, 10 days after the periodontitis model was established, the silk thread was removed for 3 days, and then the O group model was established for 10 days). That is to say, the O group represents the pure orthodontic model, the OP group represents the orthodontic model with periodontitis, and the OPL group represents the orthodontic treatment after the periodontitis treatment. The number of F4/80+, CD11b + macrophages in the gingival soft tissues around orthodontic mobile teeth (first molars) was counted using flow cytometry, and the marker CD206 was detected (Fig. 2B). The number of macrophages in the three experimental groups were lower than that in the control group. The level of inflammation (inversely correlated with the expression of CD206) in the experimental group was the highest in the OPL group and the lowest in the O group. Micro-computed tomography (CT) imaging of the maxillae of the rats in the four groups revealed a significant increase in the distance between the first and second molars in the three experimental groups following the application of the orthodontic force, indicating that the rat OTM model was successfully established (Fig. 2C). The extent of movement of the first molars in the O group was significantly greater than those in the other groups (Fig. 2C, D). On the other hand, the bone volume/ total volume (BV/TV) indices in the OP and OPL groups were lower than that in the O group (Fig. 2E); the distance of the cementoenamel junction-alveolar bone crest (CEJ-ABC) and the trabecular bone indices were not significantly different from those in the control group (Fig. 2F, G). These findings suggest that periodontitis during orthodontic treatment might result in a slight loss of bone mass and inhibit the movement of orthodontic teeth.
3. The periodontal microenvironment is an important factor in determining the rate of OTM.
The specimens were next sectioned and stained to evaluate the reasons for the differences in the rates of OTM among the four groups. New bone formation and osteoclasts numbers were observed using the hematoxylin and eosin, Masson’s, and tartrate-resistant acid phosphatase staining methods. The results showed that the amount of new bone and the number of osteoclasts in the O group were significantly higher than those in the other groups. Immunohistochemistry also showed that the expression level of the osteogenic marker Col1a1 in the O group was higher than those in the other three groups; moreover, the inflammatory markers TNF-α and IL6 were highly expressed in the OP and OPL groups (Fig. 3A, S2). Enzyme-linked immunosorbent assay showed that the bone metabolism indices in the O group were higher than those in the OP and OPL groups. Taken together, these results indicated that the OTM microenvironment in the O group was more suitable for bone remodeling (Fig. 3B, S3A).
4. CD301b + macrophages are closely related to the efficiency of OTM.
To further explain the differences in orthodontic effects in different microenvironments, we focused on the CD301b + macrophages subset. Immunofluorescence staining of the specimens showed that the number of CD301b + macrophages in the O group was significantly higher than those in the other three groups (Fig. 4A). Likewise, flow cytometry results showed that the proportion of CD301b + macrophages in the gingival soft tissues around the orthodontic mobile teeth (first molars) was significantly higher in the O group when compared to the other three groups (Fig. 4B, C). In order to further determine the relationship between CD301b + macrophages and the efficiency of OTM, orthodontic models were established after the periodontitis was relieved for 3, 7, 14, and 21 days. Flow cytometry was used to detect the number of CD301b + macrophages in the peri-gingival tissues in each group (Fig. 4D, S4). The results showed that the number of CD301b + macrophages in the periodontal tissue was highest on the 14th day of delineation and returned to normal levels on the 21st day. Furthermore, micro-CT was performed to compare the extent of movement and bone parameters in each group (Fig. 4E, F, G, H).And the longest distance of tooth movement was observed after 14 days of delineation. BV/TV analysis showed the highest values in the 7D and 14D groups, while there was no significant difference in trabecular bone data between these two groups. These findings indicated that CD301b + macrophages might be beneficial for OTM.
5. CD301b + macrophages can positively regulate the rate of OTM and the periodontal microenvironment.
The above studies have shown that CD301b + macrophages are closely related to tooth movement. Therefore, we next examined whether CD301b + macrophages contribute to tooth movement by altering the periodontitis microenvironment. CD301b + and CD301b- macrophages were sorted from primary macrophages via flow cytometry and reinfused into the periodontium on both sides of the orthodontically treated teeth in an orthodontic rat model with mild periodontitis (Fig. 5A, B). After 10 days of orthodontic treatment, the distance of movement of the teeth in the CD301b + macrophages group was significantly greater than that in the CD301b- macrophages group (Fig. 5C, D); additionally, reduced alveolar bone resorption (Fig. 5E, F) and improved periodontal conditions were observed (Fig. 5G,H) in the CD301b + macrophages group.
6. CD301b + macrophages can promote osteogenesis and enhance bone remodeling during orthodontic treatment.
To determine how CD301b + macrophages alter the periodontitis microenvironment, thereby enhancing the efficiency of OTM, we collected the culture supernatant of CD301b+/CD301b- macrophages and used them to stimulate bone marrow stromal cells (BMSCs). The cell scratch and CCK8 assays were used, and supernatants of CD301b + macrophages were found to significantly promote the migration and proliferation of BMSCs (Fig. 6A, B, C). To further mimic the in vivo orthodontic environment, we applied an appropriate amount of stretching force to the BMSCs (Fig. 6D), followed by the addition of the supernatants of the CD301b+/CD301b- macrophages culture supernatant. Alkaline phosphatase and alizarin red staining of the BMSCs revealed that the CD301b + macrophages were more effective in promoting osteogenesis (Fig. 6E). Quantitative real-time polymerase chain reaction (qRT-PCR) showed that the supernatants of CD301b + macrophages induced the expression of osteogenic differentiation-related indicators such as Runx2, Ocn, Opn, and Col1a1 in the BMSCs, which suggested that these cells might contribute to osteogenic differentiation in an orthodontic environment (Fig. 6F, S3B).